Abscopal Effect in Abscopal Effect in  p

R di i M d

Radiation Modu Radiation Modu

b l ff

Abscopal Effect in Abscopal Effect in  p

B il R R P ^{1 4} C i B é

Bertil R.R. Persson, ^1,4 Catrin Bauréus Bertil R.R. Persson, Catrin Bauréus

Crister Ceberg 1 4 ^1,4 Henrietta N Crister Ceberg, Henrietta N

1 2

1

Medical Radiation Physics,  y ,

²

Neurosurg g Lund Lund

Correspondence t Correspondence t

I d i

Introduction Introduction

We studied radiation modulated immune response in intracranial N29 tumors We studied radiation modulated immune response in intracranial N29 tumors

b bi i i l f ti f di ti th (5 ll 15 G ) d

by combining a single fraction of radiation therapy (5 as well as 15 Gy) and immunization with interferon‐gamma (IFN) transfected immunogenic tumor immunization with interferon gamma (IFN) transfected immunogenic tumor cells In a parallel study we also studied the abscopal effect of radiation therapy cells. In a parallel study we also studied the abscopal effect of radiation therapy in a model of collaterally implanted N29 tumors on both hind legs in rats, with y p g , and without the combination of immunization

and without the combination of immunization.

l d h d

Materials and Methods

Figure 1. Number of living

Materials and Methods

Figure 1. Number of living time for the rats with time for the rats with 

Animal model In two parallel studies we used

⁸

Animal model In two parallel studies, we used

⁸

inbred female and male Fischer 344 rats, inoculated

with N29 rat glioma cells The N29 cells were with N29 rat glioma cells. The N29 cells were

i l d i d f t d l d i CNS f

⁶

previously derived from tumors developed in CNS of pregnant Fischer rats exposed to ethyl‐N‐nitrosurea, pregnant Fischer rats exposed to ethyl N nitrosurea,

and have since then been successively propagated

⁴

and have since then been successively propagated

both in vitro and in vivo.

Intracranial tumors resembling human glio‐

² ₁

Intracranial tumors, resembling human glio‐

bl l if d l d i l f

¹

blastoma multiforme, were developed in a total of

44 animals after inoculation by a stereotactic

⁰

44 animals after inoculation by a stereotactic

injection of 5000 N29 cells into the head of the right

0

injection of 5000 N29 cells into the head of the right

0       50      100    

caudate nucleus.

^days aft

Extracranial tumors were induced in a total of 81 Extracranial tumors were induced in a total of 81

i l b b i l i f

^{i 2 S}

animals by a subcutaneous inoculation of 200 000

Figure 2. Set‐up

N29 cells into the right hind leg and 50 000 cells in

treatment of the e

N29 cells into the right hind leg, and 50 000 cells in th l ft hi d l

the left hind leg.

Treatment combinations The animals in the two Treatment combinations The animals in the two groups were further divided into 4 sub groups; (I) groups were further divided into 4 sub‐groups; (I) untreated controls, (II) radiotherapy alone, (III) ( ) py ( ) immunotherapy alone and (IV) radiotherapy and immunotherapy alone, and (IV) radiotherapy and

i th bi d D t th diff t

immunotherapy combined. Due to the different growth pattern and radiation response, the growth pattern and radiation response, the treatment schedule and evaluation methods treatment schedule and evaluation methods differed for the intra‐ and extracranial tumors,, respectively Sub‐optimal non‐curative dose levels respectively. Sub‐optimal, non‐curative dose levels

h f h

were chosen for each group.

Radiotherapy (RT) was given locally with collimated Radiotherapy (RT) was given locally with collimated

fields of Co 60 gamma rays Animals with intra

Figure 3. Average tu

fields of Co‐60 gamma rays. Animals with intra‐

rats with extrac

cranial tumors were treated on day 7 after the y

^{ats t e t ac}

inoculation with a single fraction of 5 Gy or 15 Gy

inoculation, with a single fraction of 5 Gy or 15 Gy.

l h l d

Animals with extracranial tumors were treated around day 30 with 4 daily fractions of 5 Gy each

around day 30, with 4 daily fractions of 5 Gy each.

I i ti (IFN ) i i t it l

Immunization (IFN

_

) was given as intraperitoneal injections of 3∙10

⁶

IFN‐gamma‐gene transfected N29 injections of 3 10 IFN gamma gene transfected N29 tumor cells The cells were given a sterilizing dose of tumor cells. The cells were given a sterilizing dose of 70 Gy just before the injections. Animals with intra‐ y j j cranial tumors were immunized 1 h after irradiation cranial tumors were immunized 1 h after irradiation

d 7 ft i l ti A i l ith t

on day 7 after inoculation. Animals with extra‐

cranial tumors were immunized 5 days before RT, cranial tumors were immunized 5 days before RT, and then two more times with 14 day intervals

and then two more times with 14‐day intervals.

Treatment evaluation For the intracranial tumors,, the effect of the treatment was evaluated in terms the effect of the treatment was evaluated in terms

f i l i Th i l b d d il f

Figure 4 Specific therapeu

of survival time. The animals were observed daily for

Figure 4. Specific therapeu abscopal effect (SAE) for ra

symptoms of the growing tumor and euthanized

abscopal effect (SAE) for ra

symptoms of the growing tumor, and euthanized

h th t b k i t t d

when the pre‐set breakpoint symptoms appeared (keeping their heads turned to one side, rotating, or

( p g , g,

losing weight) For the extracranial tumors the losing weight). For the extracranial tumors, the

ff l d f

treatment effect was evaluated in terms of tumor growth The subcutaneous tumors were estimated growth. The subcutaneous tumors were estimated

b lli id ith l th d idth d

by an ellipsoid, with length and width as measured by using a caliper. The measured tumor volume, TV, by using a caliper. The measured tumor volume, TV, was fitted by an exponential growth model was fitted by an exponential growth model,

( )

TV=TV

₀₀

∙e

^TGR∙(t‐t₀₀⁾

, where t is the time after inoculation , in days and t is the time of the first irradiation TGR in days and t

₀

is the time of the first irradiation. TGR

i th t th t t t i d

¹

Wh

is the tumor growth‐rate constant in days

^‐1

. When the tumor reached a volume of about 9 cm

³

, the the tumor reached a volume of about 9 cm , the animals were euthanized

animals were euthanized.

L d U i i / F l f S i Lund University / Faculty of Scie Lund University / Faculty of Scie

l d I R d

ulated Immune‐Response and ulated Immune‐Response and p

h l l

Rats with Contra lateral tumours Rats with Contra‐lateral tumours

K h ^{1 4} G G f ö ^{1 4} P M k f R höld ¹ s‐Koch, ^1,4  Gustav Grafström, ^1,4  Per Munck af Rosenschöld, ¹ s Koch, Gustav Grafström, Per Munck af Rosenschöld,

2 4 3 4 f f ^{2 4}

Nittby ^2,4 Bengt Widegren ^3,4 and Leif G Salford ^2,4 Nittby, Bengt Widegren, and Leif G. Salford,

3 4

gery, 

³

Tumour Immunology, 

⁴

Rausing Laboratory, Biomedical Centre, 

g y, gy, g y, ,

d University 221 85 LUND Sweden d University, 221 85 LUND, Sweden

to: Bertil R.R. Persson, bertil r.persson@med.lu.se to: Bertil R.R. Persson, bertil_r.persson@med.lu.se

C l i

Conclusions Conclusions

We found a strong enhancement of the therapeutic effect in intracranial N29 We found a strong enhancement of the therapeutic effect in intracranial N29

t i t b bi i i l f ti f di ti th d

tumors in rat by combining a single fraction of radiation therapy and immunization with IFN transfected immunogenic tumor cells. In the immunization with IFN transfected immunogenic tumor cells. In the extracranial model with collaterally implanted N29 tumors in rats we extracranial model with collaterally implanted N29 tumors in rats we demonstrated the abscopal effect of radiation therapy. In this model enhanced p py therapeutic and abscopal effect was found by radiation therapy only but no therapeutic and abscopal effect was found by radiation therapy only, but no

i ifi f d i h RT bi d i h i h

significant synergy was found with RT combined with immune‐therapy.

R l d Di i

g rats and median survival

Results and Discussion

g rats and median survival

intracranial tumours

Results and Discussion

intracranial tumours.

Intracranial tumors For the animals with intracranial Intracranial tumors For the animals with intracranial

t th b f li i i l f ti f

tumors, the number of living animals as a function of time after inoculation is shown in Figure 1 for the

153±31 (1SD) d N 8

time after inoculation is shown in Figure 1 for the rats given combined treatment with RT and

153±31 (1SD) days, N=8

rats given combined treatment with RT and immunization. The median survival times are also given in the figure The survival times of the treated given in the figure. The survival times of the treated

d h d l

groups were compared to the untreated controls,

119 35 (1SD) d N 8

and analyzed with the Mann‐Whitney test using a

119±35 (1SD) days, N=8

and analyzed with the Mann Whitney test using a

i ifi l l f 0 05 It th f d th t

significance level of =0.05. It was then found, that RT alone (5 or 15 Gy) had no significant effect on the ( y) g survival time IFN alone increased the survival time

      150      200      250

survival time. IFN

_

alone increased the survival time

h ( ) h b f

er inoculation

with 60% (p=0.04). The combination of RT at 5 Gy p y and IFN increased the survival time with 87%

f h di i

and IFN

_

increased the survival time with 87%

( 0 003) i ldi 75% l t i i

p for the radiation

(p=0.003), yielding 75% complete remissions

extracranial tumors.

(p=0.03). Also the 15 Gy RT combined with IFN

_

(p 0.03). Also the 15 Gy RT combined with IFN

_

yielded an increased survival rate although not as yielded an increased survival rate, although not as effectively as the 5 Gy RT and IFN y y

_

treatment.

Extracranial tumors For the animals with extra‐

Extracranial tumors For the animals with extra‐

i l t Fi 2 th t th t

cranial tumors, see Figure 2, the tumor growth rates (as measured by % per day) derived from the fit of (as measured by % per day) derived from the fit of the exponential growth model are displayed in the exponential growth model are displayed in Figures 3 and 4. The tumor growth rates of the g g treated groups were compared to the untreated treated groups were compared to the untreated

l d l d i h h i

controls, and analyzed with the t‐test using a significance level of =0 05 It was then found that significance level of  0.05. It was then found, that RT (20 G ) l h d i ifi t ff t th t

RT (20 Gy) alone had a significant effect on the tumor growth rate (p<0.0001), while IFN

_

alone had no

umor growth rate for

g (p ),

_

significant effect The combination of RT and IFN

cranial tumors. 

significant effect. The combination of RT and IFN

_

c a a tu o s.

also had a significant effect on the tumor growth rate g g (p=0 001) however when compared to a hypo‐

(p=0.001), however, when compared to a hypo

th ti ll dditi ff t f di th d

thetically additive effect of radiotherapy and immunization, there was no observable synergetic immunization, there was no observable synergetic effect due to the combined therapy

effect due to the combined therapy.

Comparison By treatment of single intracranial N29 p y g glioma tumors with RT in combination with glioma tumors with RT in combination with

i i i i IFN f d ll

immunization using IFN transfected tumor cells, 75% complete tumor remissions has previously been 75% complete tumor remissions has previously been demonstrated (1) That effect of radiation was demonstrated (1). That effect of radiation was related to diminishing of the tumor´s immune‐ g suppression and enhanced infiltration of activated T suppression and enhanced infiltration of activated T‐

ll ff i h h i l

cells affecting the tumor. In the present extracranial model with two contra‐lateral tumors it was

utic effect (STE) and specific

model with two contra lateral tumors it was h th i d th t ti t d T ll h ld l ff t

utic effect (STE) and specific

ts with extracranial tumors

hypothesized that activated T‐cells should also affect

ts with extracranial tumors.

the left lateral un‐irradiated tumor. The results of RT the left lateral un irradiated tumor. The results of RT alone appear as an abscopal effect with TGR alone appear as an abscopal effect with TGR decrease also in the contra‐lateral un‐irradiated tumor which account for the abscopal effect But in tumor which account for the abscopal effect. But in

thi d l i ifi t b l ff t f d

this model no significant abscopal effect was found by radiation therapy combined with immunization by radiation therapy combined with immunization using IFN transfected tumor cells This might using IFN transfected tumor cells. This might indicate that factors other than immunological are g responible for the radiation induced abscopal effect responible for the radiation induced abscopal effect.

R f

References References

1

1B Persson et al., Radiation Research 173:433‐440, 2010

/ M di l R di i Ph i

ence / Medical Radiation Physics

ence / Medical Radiation Physics